Process for the preparation of a lyophilized vaccine against duck virus hepatitis

ABSTRACT

The invention relates to a process for the preparation of the lyophilized vaccine against duck hepatitis by using the attenuated hepatitis virus TN cultivated by Asplin. 
     According to the invention the virus deposited in the Strain Collection of the Hungarian Institute of Public Healts under No. 00220 is injected into the allantoic cavity of embryonated SPF-hen&#39;s eggs, the eggs are incubated at a temperature of about 37° C., the embryos died between 24 and 96 hours are collected, homogenized with a physiological saline solution, antibiotics are added to the pure suspension, and after the addition of protective and skeleton forming agents the sterile virus material is lyophilized in a manner known per se. 
     The lyophilized vaccine can be stored at a temperature of +4° C. for one year in contradiction to the known liquid vaccine storable in freezed state at a temperature of -20° C. for half a year and which has to be used within 7 days from the delivery (with a storage temperature of +4° C.). The titre of the vaccine prepared according to the invention is at least tenfold of that of the known vaccine, thus much less embryonated eggs are used for the process according to the invention what means cost saving. Due to the lyophilization the vaccine can be transported to far destinations, too, what was not possible at the known liquid product stored in freezed state.

FIELD OF THE INVENTION

The invention relates to a process for the preparation of a lyophilizedvaccine against duck hepatitis by using the attenuated hepatitis virusTN cultivated by Asplin.

BACKGROUND OF THE INVENTION

Duck hepatitis is a dangerous viral disease to which ducklings aresusceptible in the first 4-5 weeks of their life. If they are notprotected against this disease, 40 to 50% of the young animals canperish. Therefore a protection method was sought and a vaccine suitablefor immunization was developed. For the preparation of the presentlyused vaccine the attenuated TN virus cultivated by Asplin is used(Asplin F. D. and McLauchlan J. D.: Vet. Rec. 1954, 32, 66, 456; AsplinF. D.: Vet. Rec. 1956, 68, 412). The virus whose titer has to be atleast 10⁴ EID₅₀ /ml, is injected into the allantoic cavity of 10-dayembryonated hen's egg in a dilution between 1:10 and 1:20; the eggs arecandled daily and the eggs containing deceased embryos are taken out anddestroyed in the first 24 hours. Eggs are suitable for the preparationof the vaccine in which the embryo died between 24 and 96 hours afterthe inoculation. The allantoamniotic fluid of the eggs (that is thefluid surrounding the embryo) is drawn off with vacuum, treated withantibiotics and filled into bottles.

The process was doubtlessly a great advance in the field of intensivelarge-scale poultry breeding. However the vaccine possessesdisadvantages. The main disadvantage is that it is fluid and has to bestored in frozen state at a temperature of at least -20° C. Thesubstance can be stored in this state for at most half a year. In thecourse of transport of the frozen vaccine interruptions often occur;therefore it is preferred to transport the vaccine personally to thedestination. After melting (calculated from the time of the delivery) itcan be stored at a temperature of +4° C. for 7 days. The vaccine issuitable for use if its titer reaches the value of 10³.5 EID₅₀ /ml.According to our own examinations this vaccine cannot by lyophilized,although tests were carried out with numerous protective materials. Thevirus loss caused by the lyophilization is too high, the biologicalactivity of the lyophilized substance is not sufficient.

At the production of this vaccine the material consumption is very high;for the preparation of 100 liters of vaccine, 23,000 embryonated heneggs are necessary.

OBJECTS OF THE INVENTION

The aim of the invention was to elaborate a process for producing avaccine of high titer by using less raw material, which is lyophilizableand storable for a longer time and--due to its dry state--readilytransportable.

DESCRIPTION OF THE INVENTION

In the present invention we started from the fact that the quantity ofthe virus multiplying in the embryonated hen egg is not the same at allof the different parts of the egg (Hwang J. and Dougherty E.: Avian Dis.1964, 19, 8, 264). The virus enriches mainly in the body of the chickenembryo, particularly in the liver, while just the allantoamnioticfluid--the material from which the traditional vaccine isisolated--contains the least virus.

In the face of this problem a process for the preparation of alyophilized vaccine against duck hepatitis was developed by using theattenuated hepatitis virus TN cultivated by Asplin. According to theinvention the virus deposited in the Strain Collection of the HungarianInstitute of Public Health under No. 00220 is injected into theallantoic cavity of 10-day embryonated SPF-hen eggs, the eggs areincubated at a temperature of about 37° C., the embryos died after theinoculation between the 24 and 96 hours are collected, optionally theharder parts of them are eliminated under sterile conditions, then theembryos are homogenized with physiological saline solution, centrifuged,antibiotics are added to the pure suspension and after the addition ofprotective and skeleton forming agents the virus substance isfreeze-dried by well-known methods.

The SPF (specific pathogen free) eggs for the present process must befree from Avian encephalomyelitis, fowl plague, EDS, bronchitis,leucosis A-B, Gumboro, Marek, Reovirus as well as from Salmonella Gall.,Salmonella typhi murium, Mycoplasma Gall., Mycoplasma Synoviae and theantibodies of these, respectively.

Under homogenization the suspension is advantageously diluted to such anextent that the substance consists of 25 to 35% by volume of embryo and75 to 65% by volume of physiological saline solution.

It is particularly advantageous to use a combination ofpolyvinylpyrrolidone, gelatine, glucose and saccharose as protective andskeleton forming agent.

At the lyophilization one proceeds advantageously so that the materialreceiving shelves of the freeze-drying machine are precooled to atemperature between 0° and -6° C., the substance placed on the shelvesis cooled to a temperature between -30° and -40° C., the water in thesubstance is sublimated in vacuum, after the sublimation the shelves areheated to a temperature between +30° and +40° C. and thus the substanceis dried for 4 to 10 hours.

In the process of the invention, the virus is inoculated in dilutedstate into the allantoic cavity of the 10-day embryonated SPF-egg in adose of 10,000 EID₅₀ per egg. After the inoculation the embryos deceasedwithin the first 24 hours are developed. The eggs are candled daily,collected in the 48th, 72nd and 96th hour, the eggs containing the deadembryos are stored until use at a temperature of +4° C. The front partof the embryo head (eyes, bill) is removed under sterile conditions, thebill because it is hard and the eyes because they discolour the vaccine.

The embryos are smashed in a mixer, then they are further homogenized inan ultra-homogenizer (e.g. in an ultraturrax machine) whilephysiological saline solution is added. The suspension contains about 50to 80% by volume of physiological saline solution calculated to itstotal quantity. The suspension is centrifuged with a speed of rotationof 2000-3000 minutes⁻¹ for 20 minutes, then the clear middle part isfiltered through four layers of gauze after the bone residues on thebottom of the centrifuge glass and the feather fundaments swimming onthe top of the substance have been removed. In order to examine thepurity of the filtered substance it is spread on culture-media andtreated with antibiotics (penicillin, streptomycin, neomycin andchloramphenicol). If the substance proves to be infected after a certaintime, gentamycin is added. The sterile substance is lyophilized within5-6 days from harvesting; in the meantime it is stored at +4° C.

Before the lyophilization skeleton forming and protective agents areadded to the virus suspension. E.g. the following combination isadvantageous:

5% of collidone solution (polyvinylpyrrolidone, 10% by volume)

5% of gelatine solution (10% by volume)

4% of glucose solution (50% by volume)

3% of saccharose solution (50% by volume).

The lyophilization can be carried out by well-known methods. It will bedescribed in the examples in detail.

The inoculum strain virus is prepared similarly as the vaccine but herethe sterility has to be attained to a maximum because no antibiotic canbe added to the inoculum virus.

The lyophilized vaccine can be stored at a temperature of +4° C. for oneyear.

Considering the fact that the titer of the virus suspension afterlyophilization is at least by one magnitude higher than that of thevaccine which has to be stored in freezed state, the quantity to beprepared is reduced to a tenth. Due to this fact many fewer eggs areused; note that the price of the embryonated eggs amounts to 95% of theproduction cost. At the same time a significant saving of labor isattained.

In dependence upon the virus titer, by diluting a 2 ml vial 100 to 1000single doses can be obtained according to the following relation:

    ______________________________________                                                          Necessary quantity of buffer                                        Number of for the dilution                                            titre     doses       ducklings  laying ducks                                 ______________________________________                                        10.sup.5 EID.sub.50 /ml                                                                 100         10 ml      100 ml                                       10.sup.5.3 EID.sub.50 /ml                                                               200         20 ml      200 ml                                       10.sup.5.7 EID.sub.50 /ml                                                               500         50 ml      500 ml                                       10.sup.6 EID.sub.50 /ml                                                                 1000        100 ml     1000 ml                                      ______________________________________                                    

The dose in case of both the duckling and the laying duck is at least2000 EID₅₀ that means that due to the different dilution 0.1 ml ofvaccine in the case of duckling and 1 ml of vaccine in the case oflaying duck is added. The vaccine can be administered as injectionor--in the case of ducklings, in the drinking water. It is recommendedto inoculate the breeding stock twice before the laying period. Thelaying ducks pass the immunity to the ducklings through the yolk but thethus-obtained protection lasts only for about 14 days, therefore theducklings, too, have to be treated. If the vaccine is added to thedrinking water, it is recommended to have the stock thirsted for somehours and to give the vaccinic water only after it. By the moving of theanimals it has to be ensured that every duckling can drink and suchdrinking vessels should be used in which the ducklings cannot bath.

The invention is illustrated in detail by the following examples.

EXAMPLE 1 Preparation and lyophilization of the vaccine

10-day embryonated hen's egg of SPF quality are candled, the limit ofthe air-sack and the place of the embryo are marked on the shell, thenthe blunt end of the egg is disinfected. The suspension of theegg-inoculation virus prepared with sterile physiological salinesolution is injected into the allantoic cavity through a hole made abovethe air-sack. 0.2 ml of the substance containing approximately 50,000EID₅₀ virus quantity per milliliter is used for each egg. Then the holeis closed with paraffin and the eggs are incubated in hatchers at 37° C.The embryos dying within the first 24 hours as well as those stillliving after 96 hours are destroyed. The eggs are candled daily and theeggs containing the dead embryos are stored between +2° C. and +5° C.until use.

Under sterile conditions the embryos are collected, the front part ofthe head is cut off with a pair of scissors, then the embryos aresmashed in a mixer and diluted with physiological saline solution, theyare squashed still further in an ultraturrax homogenizer. The embryosquash is further diluted while the obtained suspension contains 70% ofphysiological saline solution. This suspension is centrifuged at a speedof rotation of 3000 minutes⁻¹ for 20 minutes and the clear middle partis filtered through four layers of sterile gauze.

The thus-obtained material is submitted to a purity examination, thendirectly after the examination it is treated with antibiotics by using100,000 IU of penicillin, 760,000 IU of streptomycin, 0.2 g. ofchloramphenicol and 0.5 g of neomicyn for one liter of suspension. Thetreated suspension is allowed to stand at room temperature for one hour,then it is inoculated on culture media which is suitable for cultivationof aerob and anaerob bacteria as well as fungi. If within 3 days nogrowth of a microorganism begins on the culture media kept at roomtemperature, respectively in a thermostat, the material is consideredsterile. If it proves infected, 0.3 g. of gentamycin per liter is addedto it. The suspension is lyophilized as quickly as possible, but atleast within 5-6 days after harvesting. Until that time it is stored ata temperature of +4° C. Before the lyophilization the following sterileprotective agents are added to the suspension:

5% of collidone solution (10% by volume)

5% of gelatine solution (10% by volume)

4% of glucose solution (50% by volume)

3% of saccharose solution (50% by volume).

The total quantity of the protective and skeleton forming agents amountsto 17%. Before the lyophilization the titer of the suspension isdeterminated: 10⁷ EID₅₀ /ml.

The virus suspension is filled in doses of 2 ml into vials and is placedupon the material-receiving shelves of the freeze-drying machineprecooled to a temperature of -5° C. When the temperature of thematerial is reduced to -45° C., the vacuum pump is operated. When thepressure decreased to 0.1 mbar, the heating of the material-receivingshelves is begun and adjusted so that the temperature of the materialshould be between -30° and 35° C. (for a pressure of 0.1 mbar a shelftemperature of about -15° C. is needed). When the sublimation isfinished, the shelves are heated to a temperature of +35° C. and themaximum vacuum is put on so the material should be desiccated as much aspossible. After 8 hours drying time the water content of the product isunder a value of 2%. The vials are stoppered while still in thelyophilizer under vacuum and the rubber stopper is protected by metalstop-cap.

EXAMPLE 2 Preparation of the inoculation virus

One proceeds as in Example 1, with the difference that no antibiotic isadded to the suspension and the ready material is not lyophilized butstored in deep-frozen state in liquid nitrogen. Its titer is at least10⁶.5 -10⁷ EID₅₀ /ml. Before use a dilution is prepared from theinoculation virus in a ratio of 1:60 to 1:200; 0.2 ml of which containabout 10,000 EID₅₀ of the virus. The inoculation virus cannothaemagglutinate the erythrocytes of the hen.

EXAMPLE 3 Detection of the immune state

For lack of an appropriate virulent virus the immune state was evaluatedwith the help of a virus neutralization test carried out with serum. The480 test ducklings came from a farm where the breeding stock was notimmunized due to export interests. One group consisted of 20 testanimals.

In the vaccination tests PBS buffer of the following composition wasused for the dilution:

    ______________________________________                                        NaCl                  8       g                                               KCl                   0.2     g                                               Na.sub.2 HPO.sub.4.2H.sub.2 O                                                                       1.15    g                                               KH.sub.2 PO.sub.4     0.2     g                                               CaCl.sub.2            0.1     g                                               MgCl.sub.2.6H.sub.2 O 0.1     g                                               distilled water ad    1,000.0 ml                                              ______________________________________                                    

Different dilutions were prepared from the vaccine prepared according toExample 1 with the buffer of the above composition, and immunizationtests were carried out by injecting the dilutions subcutaneously oradded the vaccine in water. For comparison tests were carried out withthe traditional liquid vaccine, too.

12 days after the immunization blood samples were taken and the serumwas inactivated at 56° C. for 30 minutes, and the antibody level wasdetermined with the virus dilution method. The dilution of the serum ina ratio of 1:10 was mixed with the individual virus dilutions, and thethus-prepared mixtures were allowed to stand at room temperature for 1hour and then injected into 11-day embryonated SPF-eggs. The virus titreand the neutralization index (NI), respectively, were determined on thebasis of the number of embryos which died or were still living, butshowed characteristic pathological alterations after one week and it wascalculated by Reed-Muench method. The results were summarized in thefollowing table:

    ______________________________________                                                                             Difference                                                                    relative to                              Method of   Dose      Type of        the control                              treatment   EID.sub.50                                                                              administration                                                                           NI  in %                                     ______________________________________                                        control     --        --         1.6 --                                       liquid vaccine                                                                             50       subcut.    2   25                                                    613      subcut.    3   87.5                                                 6130      subcut.    3.6 125                                      lyophilized vaccine                                                                        158      subcut.    2.6 62.5                                     according to the                                                                          1585      subcut.    4.6 187.5                                    invention   15850     subcut.    4.8 200                                      lyophilized vaccine                                                                       3160      watering   3.7 131                                      according to the                                                                          31600     watering   4.4 175                                      invention   316000    watering   4.6 187.5                                    ______________________________________                                    

An index above 2 is already positive; an index above 3 is considered asa very good result. From the table it is clear that the protectiveeffect of the vaccine of the invention is excellent.

EXAMPLE 4 Proving of the storability of the vaccine

The storability of the vaccine was examined by modelling the realstorage as well as by usual thermal load. In the modelling of thestorage we started from the fact that in most countries the vaccineshave to be stored at a temperature of +4° C. according to regulations,thus the product marked for the export, too, was examined under suchconditions. The vaccine stored at a temperature between +2° and +5° C.lost 0.6 exponent (from the exponent of EID₅₀) of the activity within 12months, an irrelevant loss, but corresponds to the scattering among thevirus contents of the vials.

The thermal load test renders possible a certain fast examination. Ifthe titer of the vaccine stored at 37° C. for 1 week is stillappropriate, it is certain that it can be used for one year when storedaccording to the regulations. The vaccine prepared according to theinvention lost 0.5 exponent of activity when stored at 37° C. for 1 weekwhat is a very advantageous result.

We claim:
 1. A process for the preparation of a lyophilized vaccineagainst duck hepatitis comprising the steps of:(a) injecting a hepatitisvirus TN as deposited in the Strain Collection of the HungarianInstitute of Public Health under No. 00220, into the allantoic cavity ofembryonated SPF hen eggs; (b) incubating the eggs at a temperature ofabout 37° C.; (c) collecting the embryos dying between 24 and 96 hoursfrom the start of incubation; (d) discarding the embryos dying prior to24 and subsequent to 96 hours form the start of incubation; (e)homogenizing the collected embryos in physiological saline solution toform a suspension; (f) centrifuging the suspension; (g) adding to thesuspension an antibiotic selected from the group consisting ofpenicillin, streptomycin, chloramphenicol, neomycin, gentamycin, andmixtures thereof in an amount sufficient to sterilize said suspension,and adding to the suspension a protective skeleton-forming agentconsisting essentially of:5% of collidone solution, 5% of gelatinsolution, 4% of glucose solution, and 3% of sucrose solution, so thatthe total quantity of the protective skeleton-forming agents in thesuspension is 17% by weight; and (h) lyophilizing the resultingcomposition.
 2. A lyophilized vaccine against duck hepatitis made by thesteps of:(a) injecting a hepatitis virus TN as deposited in the straincollection of the Hungarian Institute of Public Health under No. 00220,into the allantoic cavity of embryonated SPF hen eggs; (b) incubatingthe eggs at a temperature of about 37° C.; (c) collecting the embryosdying between 24 and 96 hours from the start of incubation; (d)discarding the embryos dying prior to 24 and subsequent to 96 hours fromthe start of incubation; (e) homogenizing the collected embryos inphysiological saline solution to form a suspension; (f) centrifuging thesuspension; (g) adding to the suspension an antibiotic selected from thegroup consisting of penicillin, streptomycin, chloramphenicol, neomycin,gentamycin, and mixtures thereof in an amount sufficient to sterilizesaid suspension, and adding to the suspension a protectiveskeleton-forming agent consisting essentially of:5% of collidonesolution, 5% of gelatin solution, 4% of glucose solution, and 3% ofsucrose solution, so that the total quantity of the protectiveskeleton-forming agents in the suspension is 17% by weight; and (h)lyophilizing the resulting composition.
 3. A method of incubating ducksagainst duck hepatitis which comprises administering to the ducks aneffective amount of the vaccine prepared by the steps of:(a) injecting ahepatitis virus TN as deposited in the strain collection of theHungarian Institute of Public Health under No. 00220, into the allantoiccavity of embryonated SPF hen eggs; (b) incubating the eggs at atemperature of about 37° C.; (c) collecting the embryos dying between 24and 96 hours from the start of incubation; (d) discarding the embryosdying prior to 24 and subsequent to 96 hours from the start ofincubation; (e) homogenizing the collected embryos in physiologicalsaline solution to form a suspension; (f) centrifuging the suspension;(g) adding to the suspension an antibiotic selected from the groupconsisting of penicillin, streptomycin, chloramphenicol, neomycin,gentamycin, and mixtures thereof in an amount sufficient to sterilizesaid suspension, and adding to the suspension, a protectiveskeleton-forming agent consisting essentially of:5% of collidonesolution; 5% of gelatin solution, 4% of glucose solution, 3% of sucrosesolution, so that the total quantity of the protective skeleton-formingagents in the suspension is 17% by weight; and (h) lyophilizing theresulting composition.